Vasoactive agonists and growth factors activate multiple signalling pathways in vascular smooth muscle, including activation of phospholipase C, stimulation of tyrosine kinases, and activation of phospholipase D. In cultured vascular smooth muscle cells, we have found that the single most robust stimulation of any signalling pathway is activation of phospholipase D. In contrast, stimulation of this pathway in normal arteries is minimal. More recently, we have shown that balloon injury of carotid arteries causes a marked, transient activation of phospholipase D that precedes the proliferative response. Taken together, these results suggest that phospholipase D may be intimately related to the growth program in vascular smooth muscle. Strategies to test this hypothesis have been limited by the lack of structural information about or analytical reagents with which to study the function of phospholipase D. Therefore, we propose to isolate, characterize and clone the vascular smooth muscle phospholipase D in order to provide tools with which to probe its role in vascular smooth muscle cell physiology. Once these probes are obtained, we will study the expression of phospholipase D mRNA and protein, as well as activity, in differentiated and proliferating vascular smooth muscle, and will begin to define the growth-related factors which control its expression and activity. These studies will provide new insight into the physiologic role of phospholipase D activation, and will ultimately contribute to our understanding of initiation of the vascular smooth muscle cell proliferation that is an integral event in atherogenesis and restenosis after angioplasty.